PROJECT SUMMARY Arrestin proteins are master regulators of G protein coupled receptor (GPCR) signaling, and act in two ways. First, arrestins terminate the coupling of G proteins to cognate receptor by physically blocking the G protein coupling site. Second, arrestins can support G protein independent signaling. The best studied arrestin- mediated signaling pathways include the activation of mitogen activated protein (MAP) kinases. Recently we have determined the structure of activated arrestin-3, which shows the expected inter-domain twist. We also identified localized, previously unrecognized conformational changes in the effector binding regions of activated arrestin. Combining structural analysis with functional measurements, we propose a new paradigm for arrestin scaffolding of effectors via these sites, where the transition of arrestin into the active conformation can ?turn on? effector binding. Functionally analogous to `switch regions' of G proteins, these activation- dependent conformational changes in effector binding regions are 30-40 distal from the site of receptor (or non-receptor activator) binding and are distinct from activation-associated conformational changes previously described in the literature. We leverage this new finding with three aims that explore the connection between activation and effector binding in three aims. In Aim 1, we combine in vitro structural and biophysical techniques (X-ray crystallography, multi-angle laser light scattering (MALLS), and affinity measurements, with double electron electron resonance (DEER) as an alternative approach) with single molecule and functional measurements in cells to investigate the role of oligomerization in stabilizing receptor-independent arrestin activation. In Aim 2, we combine mutagenesis and functional analysis to reveal how arrestin-phosphate and arrestin- protein interactions at the activation sites of arrestin is allosterically transmitted to the effector binding sites, which are 30 ? 40 distal. In Aim 3, we use new methods to stabilize the active form of arrestin-3 and use X-ray crystallography to identify the details of arrestin-effector interactions.